Iq sybr real time kit

Total RNA was extracted with Trizol reagent (Invitrogen). Oligo (dT)₁₈ Primer and M-MLV Reverse Transcriptase (Invitrogen) were used to generate cDNA. Gene expression was examined with the iQ SYBR real-time kit (Bio-Rad Laboratories). Data were normalized using beta-actin (Actb) as the reference gene. The primer pairs for qRT–PCR analysis of Il2, Ifng, Cblb and Tbx21 were previously described^{18 (link)}. Primer pairs for detection of mouse Nr4a1, Itch, Klf4, Hivep3 and Lpin2 were as follows: Nr4a1, forward, 5′-AGTTGGGGGAGTGTGCTAGA-3′; reverse, 5′-GCTTGAATACAGGGCATCTCCAG-3′. Itch, forward, 5′-ACCCT AAAGTTGTGGGGATGG-3′; reverse, 5′-GTTTGGCTGAGATGACAGTGA-3′. Klf4, forward, 5′-GACTAACCGTTGGCGTGAGG-3′; reverse, 5′-GTCTAGGTCCAGGAGGTCGT-3′. Hivep3, forward, 5′-CCCCCTA GTTCTC TGCCTCT-3′; reverse, 5′-GCTTAGATCGCAGGAGGGAC-3′. Lpin2, forward, 5′-TGTTTGTGGTGCCTTCACCT-3′; reverse, 5′-TGGTTTGAGA CAGAATCCCAGG-3′.

Total RNA was extracted with Trizol reagent (Invitrogen). Oligo (dT), and MMLV reverse transcriptase (Invitrogen) were used to generate cDNA. Gene expressions were examined by iQ™ SYBR real-time kit (Bio-Rad Laboratories). The data were normalized to a reference gene beta-actin. The primer pairs for real time RT-PCR analysis of Bcl6, Ascl2, CXCR5, Blimp1, Gata3, T-bet, RORγ, Tet1, Tet2, and Tet3 were previously described (Ichiyama et al., 2015 (link); Liu et al., 2012 (link)). Primer pair for detection of mouse Tcf7 is: forward, 5′-GGGAAAAACATCAAGAATCCACCA-3′; reverse, 5′-CTGGGCTAGCAAGCAGTTCT-3′.

Reactive tonsils were obtained from children undergoing elective tonsillectomy after informed consent on an institutional review board-approved protocol ²⁰ . Total RNA was extracted with Trizol reagent (Invitrogen). Oligo (dT), and MMLV reverse transcriptase (Invitrogen) were used to generate cDNA. Gene expressions were examined by iQ^™ SYBR real-time kit (Bio-Rad Laboratories). The data were normalized to a reference gene beta-actin. The primer pairs for real time RT-PCR analysis of Bcl6, Batf, CXCR5, IL-21, IL-4, Gata3, IFN-γ, T-bet, Il-17A, and RORγ were previously described ^{4 (link)}. Primer pair for detection of mouse Ascl2 is: forward, 5′-CGCTGCCCAGACTCATGCCC-3′; reverse, 5′-GCTTTACGCGGTTGCGCTCG -3′.